This protocol can be used to produce time-lapse movies of Pseudomonas aeruginosa swarming and to capture the repulsion of swarm by bacteriophage or antibiotics. The time-lapse movies capture the detailed dynamics of the swarming response to antibiotics and bacteriophage and the technique only requires a dehumidifier, computer, incubator, and document scanner. To prepare swarming agar plates for P.aeruginosa swarming time-lapse imaging, use a 25 milliliter pipette to add 20 milliliters of swarming agar solution to each experimental 10 centimeter diameter Petri dish.
Place the plates in a single stack with lids for one hour at room temperature. Turn on a dehumidifier to decrease the relative humidity of the room to 40-50%When the agar has solidified, dry the swarming agar plates for an additional 30 minutes in a laminar flow hood with the lids off and face up at 300 cubic feet per minute with a 40-50%relative humidity at room temperature. Then store the dried swarming agar plates at four degrees Celsius for up to 24 hours.
For swarming agar plating, hold a P20 pipette tip loaded with five microliters from an overnight P.aeruginosa culture at a 10-45 degree angle 2-1/2 centimeters above the center of one swarming plate and depress the plunger to the first stop touching the agar with only the liquid drop. Use a template to position the spot consistently across the different swarming agar plates. To plate a phage infection, first mix 30 microliters of overnight cultured P.aeruginosa with six microliters of one times 10 to the 12 plaque-forming units per milliliter of phage DMS3 vir and immediately add six microliters of the P.aeruginosa phage mixture at six equidistant satellite positions on a 2.8 centimeter radius concentric circle as demonstrated.
Use a template to position the spot consistently across different swarming agar plates. To plate antibiotic treatments, mix 30 microliters of overnight cultured P.aeruginosa with the appropriate concentration and volume of the antibiotic of interest and immediately add six microliters of the antibiotic-treated bacteria at six equidistant satellite positions on a 2.8 centimeter radius concentric circle about the center of the dish. Use a template to position the spot consistently across the different swarming agar plates.
When all of the cultures have been plated, replace the clear Petri dish lids with custom-made black lids. Carefully place the swarming agar plates on a scanner in an incubator set at 37 degrees Celsius with a 10 liter water bath to maintain the humidity at 75%After placing the plates into the scanner, open file and save settings to set the saving path for the images and click scan and OK at 30-minute intervals. To automate the image acquisition, use the scripting software.
Select both idle scanning and single scan and right-click on idle scanning, then left-click on enabled. At the end of the incubation, import all of the scanned images into ImageJ. In the sequence options window, number of images will indicate the number of images selected.
Keep starting image at one to start from the first picture in the folder and the scale images at 100%to conserve the original size of the images. Click covert to RGB to keep the images in color. Leave use virtual stack unchecked and click OK to load the images.
Click file, save as and AVI to merge the images into an AVI file. Adjust the compression to JPEG and the frame rate to five frames per second. Then save the AVI time-lapse in the appropriate storage folder.
Time-lapse imaging of a single colony of wild-type P.aeruginosa from an LB agar plate in LB broth overnight at 37 degrees Celsius and spotted into the center of a swarming agar plate reveals an initial growth in the form of a colony at the center of the plate that spreads in tendrils radially from the colony. Swarms move from the center of the swarming agar plates to the periphery and are repelled by a stress signal emitted by the bacteria that were infected with phages or treated with Gentamycin. Phages or Gentamycin spotted alone at the satellite positions do not cause swarming populations to avoid these areas.
Use a 25 milliliter pipette to ensure that the swarming agar plates contain exactly 20 milliliters. It is also important to adjust the plate drying time according to the laboratory environment. Our technique enables us to visualize the overall swarming patterns of Pseudomonas aeruginosa populations.
The next objective is to monitor individual cell motility using Brightfield microscopy.
